A visible light-crosslinkable decellularized kidney matrix bioink for 3D bioprinting of organoids and drug testing

Abstract

Decellularized kidney extracellular matrix (DKM) is an acellular scaffold rich in structural proteins and glycosaminoglycans that can promote tissue regeneration and support organoid culture. Porcine-derived DKM contains abundant extracellular matrix (ECM) components, such as collagen, laminin, and fibronectin, and offers native biochemical cues. However, conventional decellularized ECM hydrogels often exhibit weak mechanical properties, poor printability, and slow gelation, limiting their use in high-throughput applications. Here, we report a visible-light-mediated crosslinking strategy for rapid gelation of DKM based on a tris(2,2’-bipyridyl) ruthenium (II) chloride hexahydrate/sodium persulfate (Ru/SPS) photoinitiator system. Illumination at 405 nm (30 mW/cm2) in the presence of Ru/SPS achieves gelation in about 40 s, yielding a composite DKM–Ru/SPS bioink with tunable modulus by adjusting DKM, Ru, and SPS concentrations. High-fidelity 3D constructs were produced by extrusion bioprinting using a representative formulation (15 mg/mL DKM, 0.25 mM Ru, 2.5 mM SPS). As proof of concept, organoids encapsulated in the DKM–Ru/SPS bioink exhibited viability, proliferation, and lineage marker expression during culture. This work demonstrates a rapid, cell-compatible photocrosslinking approach for DKM–Ru/SPS that integrates organoid culture with 3D bioprinting and drug testing, supporting its potential use as a standardized bioink in tissue engineering and functional screening.

 

Keywords

3D bioprinting / Decellularized kidney matrix / Drug screening / Organoid culture / Visible-light-crosslinkable

Funding

The authors acknowledge the financial support from the National Key Research and Development Program of China (Nos. 2022YFA1105200 and 2022YFB3804700), the Central Government Guiding Local Science and Technology Development Special Fund Projects in Sichuan Province (No. 2023ZYD0166), the Chengdu City Science and Technology Project of “Unveiling and Commanding” (No. 2024-JB00-00018-GX), and the Eastern New District of Chengdu for the Implementation of Technological Innovation Projects (No. 2024-DBXQ-KJYF002).

Conflict of Interest

The authors declare no conflicts of interest.

References

[1] Frankell AM, Dietzen M, Al Bakir M, et al. The evolution of lung cancer and impact of subclonal selection in TRACERx. Nature. 2023; 616(7957):525-533. doi: 10.1038/s41586-023-05783-5

[2] Lian L, Xie M, Luo Z, et al. Rapid volumetric bioprinting of decellularized extracellular matrix bioinks. Adv Mater. 2024; 36(34):2304846. doi: 10.1002/adma.202304846

[3] Kratochvil MJ, Seymour AJ, Li TL, Paşca SP, Kuo CJ, Heilshorn SC. Engineered materials for organoid systems. Nat Rev Mater. 2019; 4(9):606-622. doi: 10.1038/s41578-019-0129-9

[4] Xie M, Sun Y, Wang J, et al. Thermo-sensitive sacrificial microsphere-based bioink for centimeter-scale tissue with angiogenesis. IJB. 2022; 8(4):599. doi: 10.18063/ijb.v8i4.599

[5] Liu J, Wu G, Wu D, et al. Microfluidic organoid-slice-on-a-chip system for studying anti-cholangiocarcinoma drug efficacy and hepatorenal toxicity. Lab Chip. 2025; 25(12): 2839-2850. doi: 10.1039/D4LC00902A

[6] Wu G, Wu D, Hu W, et al. A novel microfluidic self-perfusion chip (MSPC) for pumpless 3D cell, microtissue and organoid culture. Lab Chip. 2025; 25(15):3694-3706. doi: 10.1039/D5LC00030K

[7] Hu W, Bei HP, Jiang H, et al. DLM-GelMA/tumor slice sandwich structured tumor on a chip for drug efficacy testing. Lab Chip. 2024; 24(15):3718-3727. doi: 10.1039/D4LC00278D

[8] Wu M, Huang K, Long C, Yang C, Tong Y. In vitro extracellular matrix model to evaluate stroma cell response to transvaginal mesh. Neurourol Urodyn. 2013; 33(4):449-454. doi: 10.1002/nau.22425

[9] Song H, Jiang H, Hu W, et al. Cervical extracellular matrix hydrogel optimizes tumor heterogeneity of cervical squamous cell carcinoma organoids. Sci Adv. 2024; 10(20):eadl3511. doi: 10.1126/sciadv.adl3511

[10] Kim J, Kim J, Gao G, et al. Bioprinted organoids platform with tumor vasculature for implementing precision personalized medicine targeted towards gastric cancer. Adv Funct Mater. 2023; 34(11):2306676. doi: 10.1002/adfm.202306676

[11] Treacy NJ, Clerkin S, Davis JL, et al. Growth and differentiation of human induced pluripotent stem cell (hiPSC)-derived kidney organoids using fully synthetic peptide hydrogels. Bioact Mater. 2023; 21:142-156. doi: 10.1016/j.bioactmat.2022.08.003

[12] Lin L, Jiang S, Yang J, et al. Application of 3D-bioprinted nanocellulose and cellulose derivative-based bio-inks in bone and cartilage tissue engineering. IJB. 2022; 9(1):637. doi: 10.18063/ijb.v9i1.637

[13] Garreta E, Moya-Rull D, Marco A, et al. Natural hydrogels support kidney organoid generation and promote in vitro angiogenesis. Adv Mater. 2024; 36(34):2400306. doi: 10.1002/adma.202400306

[14] Jin Y, Zhang J, Xu Y, et al. Stem cell-derived hepatocyte therapy using versatile biomimetic nanozyme incorporated nanofiber-reinforced decellularized extracellular matrix hydrogels for the treatment of acute liver failure. Bioact Mater. 2023; 28:112-131. doi: 10.1016/j.bioactmat.2023.05.001

[15] Wei Q, Liu D, Chu G, et al. TGF-β1-supplemented decellularized annulus fibrosus matrix hydrogels promote annulus fibrosus repair. Bioact Mater. 2023; 19:581-593. doi: j.bioactmat.2022.04.025

[16] Zhang X, Chen X, Hong H, Hu R, Liu J, Liu C.Decellularized extracellular matrix scaffolds: Recent trends and emerging strategies in tissue engineering. Bioact Mater. 2022; 10:15-31. doi: j.bioactmat.2021.09.014

[17] Lu J, Huang J, Jin J, Xie C, Xue B, Lai J. The design and characterization of a strong Bio-Ink for meniscus regeneration. IJB. 2022; 8(4):600. doi: 10.18063/ijb.v8i4.600

[18] Nerger BA, Sinha S, Lee NN, et al. 3D hydrogel encapsulation regulates nephrogenesis in kidney organoids. Adv Mater. 2024; 36(14):2308325. doi: 10.1002/adma.202308325

[19] Lee J, Hong J, Kim W, Kim GH. Bone-derived dECM/alginate bioink for fabricating a 3D cell-laden mesh structure for bone tissue engineering. Carbohydr Polym. 2020; 250:116914. doi: j.carbpol.2020.116914

[20] Rizzo R, Ruetsche D, Liu H, Zenobi‐Wong M. Optimized photoclick (bio)resins for fast volumetric bioprinting. Adv Mater. 2021; 33(49):2102900. doi: 10.1002/adma.202102900

[21] Chen S, Wu C, Zhou T, et al. Aldehyde-methacrylatehyaluronan profited hydrogel system integrating aligned and viscoelastic cues for neurogenesis. Carbohydr Polym. 2022; 278:118961.

[22] Milton LA, Davern JW, Hipwood L, et al. Liver click dECM hydrogels for engineering hepatic microenvironments. Acta Biomater. 2024; 185:144-160. doi: 10.1016/j.actbio.2024.06.037

[23] Elvitigala KCML, Mohan L, Mubarok W, Sakai S. Phototuning of hyaluronic‐acid‐based hydrogel properties to control network formation in human vascular endothelial cells. Adv Healthc Mater. 2024; 13(17):2303787. doi: 10.1002/adhm.202303787

[24] Lim KS, Schon BS, Mekhileri NV, et al. New visible-light photoinitiating system for improved print fidelity in gelatin-based bioinks. ACS Biomater Sci Eng. 2016; 2(10):1752-1762. doi: 10.1021/acsbiomaterials.6b00149

[25] Kim H, Kang B, Cui X, et al. Light‐activated decellularized extracellular matrix‐based bioinks for volumetric tissue analogs at the centimeter scale. Adv Funct Mater. 2021; 31(32):2011252. doi: 10.1002/adfm.202011252

[26] Wang L, Riediger L, Rao Q, et al. Tunable synthetic hydrogel modulates hepatic lineage specification of human liver organoid. Adv Funct Mater. 2025; 19(8):e08430. doi: 10.1002/adfm.202508430

[27] Fang W, Yang M, Wang L, et al. Hydrogels for 3D bioprinting in tissue engineering and regenerative medicine: current progress and challenges. IJB. 2023; 9(5):759. doi: 10.18063/ijb.759

[28] Wang Z, Boretto M, Millen R, et al. Rapid tissue prototyping with micro-organospheres. Stem Cell Rep. 2022; 17(9):1959-1975. doi: 10.1016/j.stemcr.2022.07.016

[29] Liu F, Liu C, Chen Q, et al. Progress in organ 3D bioprinting. IJB. 2024; 4(1):128. doi: 10.18063/ijb.v4i1.128

[30] Hu Y, Zhu T, Cui H, Cui H. Integrating 3D bioprinting and organoids to better recapitulate the complexity of cellular microenvironments for tissue engineering. Adv Healthc Mater. 2024; 14(3):2403762. doi: 10.1002/adhm.202403762

[31] Choi Y mi, Lee H, Ann M, Song M, Rheey J, Jang J. 3D bioprinted vascularized lung cancer organoid models with underlying disease capable of more precise drug evaluation. Biofabrication. 2023; 15(3):034104. doi: 10.1088/1758-5090/acd95f

[32] Han Y, Yang J, Zhao W, et al. Biomimetic injectable hydrogel microspheres with enhanced lubrication and controllable drug release for the treatment of osteoarthritis. Bioact. Mater. 2021; 6(10):3596-3607. doi: 10.1016/j.bioactmat.2021.03.022

[33] Rawal P, Tripathi DM, Ramakrishna S, Kaur S. Prospects for 3D bioprinting of organoids. Bio-Des Manuf. 2021; 4(3):627-640. doi: 10.1007/s42242-020-00124-1

[34] Alam K, Nair L, Mukherjee S, et al. Cellular interplay to 3D in vitro microphysiological disease model: cell patterning microbiota-gut-brain axis. Bio-Des Manuf. 2024; 7(3):320-357. doi: 10.1007/s42242-024-00282-6

[35] Keselowsky BG, Bridges AW, Burns KL, et al. Role of plasma fibronectin in the foreign body response to biomaterials. Biomaterials. 2007; 28(25):3626-3631. doi: j.biomaterials.2007.04.035

[36] Xie M, Lian L, Mu X, et al. Volumetric additive manufacturing of pristine silk-based (bio)inks. Nat Commun. 2023; 14(1):210. doi: 10.1038/s41467-023-35807-7

[37] Yang H, Zeng T, Jiang T, et al. Recent progress on functional hydrogels as biomaterial for biomedicine. Results Chem. 2025; 16:102464. doi: 10.1016/j.rechem.2025.102464

[38] Datta P, Barui A, Wu Y, Ozbolat V, Moncal KK, Ozbolat IT. Essential steps in bioprinting: from pre- to post-bioprinting. Biotechnol Adv. 2018; 36(5):1481-1504. doi: 10.1016/j.biotechadv.2018.06.003

[39] Luo Y, He X, Du Q, et al. Metal‐based smart nanosystems in cancer immunotherapy. Exploration. 2024; 4(6):20230134. doi: 10.1002/EXP.20230134

[40] Chen B, Guo K, Zhao X, et al. Tumor microenvironmentresponsive delivery nanosystems reverse immunosuppression for enhanced CO gas/immunotherapy. Exploration. 2023; 3(6):20220140. doi: 10.1002/EXP.20220140